Process for bacterial production of polypeptides

ABSTRACT

Refractile particles containing a heterologous polypeptide as an insoluble aggregate are recovered from bacterial periplasm. The process involves culturing bacterial cells so as to express nucleic acid encoding phage lysozyme and nucleic acid encoding the heterologous polypeptide under separate promoters, disrupting the cells mechanically to release the phage lysozyme so as to release refractile particles from the bacterial cellular matrix, and recovering the released refractile particles from the periplasm. Chloroform is not used in any step and the recovery step minimizes co-recovery of cellular debris with the released refractile particles.

RELATED APPLICATIONS

This application is a non-provisional application filed under 37 CFR1.53(b) (1), claiming priority under 35 USC 119(e) to provisionalapplication No. 60/106,053 filed 28 Oct. 1998, the contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a process for producing and recoveringpolypeptides from bacterial cells. More particularly, this inventionrelates to a process wherein recovery of insoluble recombinantpolypeptides from bacterial periplasm is increased.

2. Description of Related Disclosures

Escherichia coli has been widely used for the production of heterologousproteins in the laboratory and industry. E. coli does not generallyexcrete proteins to the extracellular medium apart from colicins andhemolysin (Pugsley and Schwartz, Microbiology, 32: 3-38 (1985)).Heterologous proteins expressed by E. coli may accumulate as solubleproduct or insoluble aggregates. See FIG. 1 herein. They may be foundintracellularly in the cytoplasm or be secreted into the periplasm ifpreceded by a signal sequence. How one proceeds initially in therecovery of the products greatly depends upon how and where the productaccumulates. Generally, to isolate the proteins, the cells may besubjected to treatments for periplasmic extraction or be disintegratedto release trapped products that are otherwise inaccessible.

The secretion of recombinant proteins to the periplasmic space hasnumerous advantages over expression in the cytoplasm. The periplasmicspace contains only 7 out of the 25 known cellular proteases (Swamy andGoldberg, J. Bacteriol., 149: 1027-1033 (1982); French and Ward, J.Chem. Tech. and Biol., 54 (3): 301 (1992)) and comprises only 4-8% ofthe total cell protein (Beacham, Int. J. Biochem., 10: 877-883 (1979)).The mature secreted protein does not include N-formyl methionine and theoxidative environment of the periplasm facilitates correct disulfidebonding and protein folding (Fahey et al., J. Mol. Evol., 10: 155-160(1977)). Numerous heterologous proteins have been secreted to theperiplasmic space of E. coli. Some have involved use of fusion proteins(Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA, 75: 3727-3731(1978); EP 6,694; and U.S. Pat. No. 4,336,336). Specific productsprepared include antibody fragments (Pluckthun, Nature, 347: 497-498(1990); WO 93/06217), ribonuclease A (Tarragona-Fiol et al., Gene, 118:239-245 (1992)), HIV-1 receptor (Rochenbach et al., Appl. Microbiol.Biotechnol., 35: 32-37 (1991)), trypsin (Vasquez et al., J. Cell.Biochem., 39: 265-276 (1989)); human stefin A (Strauss et al., Biol.Chem. Hoppe Syeler, 369: 1019-1030 (1988)), xylanase (Bon-Joon et al.,J. Microb. and Tech., 6: 414-419 (1996)), rat GM-CSF (Holowachuk andRuhoff, Protein Exp. and Purification, 6: 588-596 (1995)), andinterleukin-2 (Halfmann et al., J. Gen. Microbiol., 139: 2465-2473(1993)).

The conventional isolation of heterologous polypeptide fromgram-negative bacteria poses problems owing to the tough, rigid cellwalls that surround these cells. The bacterial cell wall maintains theshape of the cell and protects the cytoplasm from osmotic pressures thatmay cause cell lysis; it performs these functions as a result of ahighly cross-linked peptidoglycan (also known as murein) backbone thatgives the wall its characteristic rigidity. A recent model described thespace between the cytoplasmic and outer membranes as a continuous phasefilled with an inner periplasmic polysaccharide gel that extends into anouter highly cross-linked peptidoglycan gel (Hobot et al., J. Bact.,160: 143 (1984)). This peptidoglycan sacculus constitutes a barrier tothe recovery of any heterologous polypeptide not excreted by thebacterium into the medium.

To release recombinant proteins from the E. coli periplasm, treatmentsinvolving chemicals such as chloroform (Ames et al., J. Bacteriol., 160:1181-1183 (1984)), guanidine-HCl, and Triton X-100 (Naglak and Wang,Enzyme Microb. Technol., 12: 603-611 (1990)) have been used. However,these chemicals are not inert and may have detrimental effects on manyrecombinant protein products or subsequent purification procedures.Glycine treatment of E. coli cells, causing permeabilization of theouter membrane, has also been reported to release the periplasmiccontents (Ariga et al., J. Ferm. Bioeng., 68: 243-246 (1989)). Thesesmall-scale periplasmic release methods have been designed for specificsystems. They do not translate easily and efficiently and are generallyunsuitable as large-scale methods.

The most widely used methods of periplasmic release of recombinantprotein are osmotic shock (Nosal and Heppel, J. Biol. Chem., 241:3055-3062 (1966); Neu and Heppel, J. Biol. Chem., 240: 3685-3692(1965)), hen eggwhite (HEW)-lysozyme/ethylenediamine tetraacetic acid(EDTA) treatment (Neu and Heppel, J. Biol. Chem., 239: 3893-3900 (1964);Witholt et al., Biochim. Biophys. Acta, 443: 534-544 (1976); Pierce etal., ICheme Research Event, 2: 995-997 (1995)), and combinedHEW-lysozyme/osmotic shock treatment (Erench et al., Enzyme and Microb.Tech., 19: 332-338 (1996)). Typically, these procedures include aninitial disruption in osmotically-stabilizing medium followed byselective release in non-stabilizing medium. The composition of thesemedia (pH, protective agent) and the disruption methods used(chloroform, HEW-lysozyme, EDTA, sonication) vary among specificprocedures reported. A variation on the HEW-lysozyme/EDTA treatmentusing a dipolar ionic detergent in place of EDTA is discussed by Stabelet al., Veterinary Microbiol., 38: 307-314 (1994). For a general reviewof use of intracellular lytic enzyme systems to disrupt E. coli, seeDabora and Cooney in Advances in Biochemical Engineering/Biotechnology,Vol. 43, A. Fiechter, ed. (Springer-Verlag: Berlin, 1990), pp. 11-30.

HEW-lysozyme acts biochemically to hydrolyze the peptidoglycan backboneof the cell wall. The method was first developed by Zinder and Arndt,Proc. Natl. Acad. Sci. USA, 42: 586-590 (1956), who treated E. coli withegg albumin (which contains HEW-lysozyme) to produce rounded cellularspheres later known as spheroplasts. These structures retained somecell-wall components but had large surface areas in which thecytoplasmic membrane was exposed.

U.S. Pat. No. 5,169,772 discloses a method for purifying heparinase frombacteria comprising disrupting the envelope of the bacteria in anosmotically-stabilized medium, e.g., 20% sucrose solution using, e.g.,EDTA, lysozyme, or an organic compound, releasing thenon-heparinase-like proteins from the periplasmic space of the disruptedbacteria by exposing the bacteria to a low-ionic-strength buffer, andreleasing the heparinase-like proteins by exposing thelow-ionic-strength-washed bacteria to a buffered salt solution.

There are several disadvantages to the use of the HEW-lysozyme additionfor isolating periplasmic proteins. The cells must be treated with EDTA,detergent, or high pH, all of which aid in weakening the cells. Also,the method is not suitable for lysis of large amounts of cells becausethe lysozyme addition is inefficient and there is difficulty indispersing the enzyme throughout a large pellet of cells.

Many different modifications of these methods have been used on a widerange of expression systems with varying degrees of success(Joseph-Liazun et al., Gene, 86: 291-295 (1990); Carter et al.,Bio/Technology, 10: 163-167 (1992)). Although these methods have workedon a laboratory scale, they involve too many steps for an efficientlarge-scale recovery process.

Efforts to induce recombinant cell culture to produce lysozyme have beenreported. EP 155,189 discloses a means for inducing a recombinant cellculture to produce lysozymes, which would ordinarily be expected to killsuch host cells by means of destroying or lysing the cell wallstructure. Russian Pat. Nos. 2043415, 2071503, and 2071501 discloseplasmids and corresponding strains for producing recombinant proteinsand purifying water-insoluble protein agglomerates involving thelysozyme gene. Specifically, the use of an operon consisting of thelysozyme gene and a gene that codes for recombinant protein enablesconcurrent synthesis of the recombinant protein and a lysozyme thatbreaks the polysaccharide membrane of E. coli.

U.S. Pat. No. 4,595,658 discloses a method for facilitatingexternalization of proteins transported to the periplasmic space of E.coli. This method allows selective isolation of proteins that locate inthe periplasm without the need for lysozyme treatment, mechanicalgrinding, or osmotic shock treatment of cells. U.S. Pat. No. 4,637,980discloses producing a bacterial product by transforming atemperature-sensitive lysogen with a DNA molecule that codes, directlyor indirectly, for the product, culturing the transformant underpermissive conditions to express the gene product intracellularly, andexternalizing the product by raising the temperature to inducephage-encoded functions. JP 61-257931 published Nov. 15, 1986 disclosesa method for recovering IL-2 using HEW-lysozyme. Asami et al., J.Ferment. and Bioeng., 83: 511-516 (1997) discloses synchronizeddisruption of E. coli cells by T4 phage infection, and Tanji et al., J.Ferment. and Bioeng., 85: 74-78 (1998) discloses controlled expressionof lysis genes encoded in T4 phage for the gentle disruption of E. colicells.

The development of an enzymatic release method to recover recombinantperiplasmic proteins suitable for large-scale use is reported by Frenchet al., Enzyme and Microbial Technology, 19: 332-338 (1996). This methodinvolves resuspension of the cells in a fractionation buffer followed byrecovery of the periplasmic fraction, where osmotic shock immediatelyfollows lysozyme treatment. The effects of overexpression of therecombinant protein, S. thermoviolaceus α-amylase, and the growth phaseof the host organism on the recovery are also discussed.

Further, E. coli mutants that leak various periplasmic enzymes have beenisolated. For example, Lopes et al., J. Bacteriol., 109(2): 520-525(1972) treated E. coli cells with a mutagen such as nitrosoguanidine,and mutants excreting periplasmic enzymes were selected by enzyme assaysystems. Such mutants included those leaking ribonuclease I,endonuclease I, and alkaline phosphatase. It is believed that thesemutants are deficient in some component of the outer bacterial membraneleading to an increase in the cells' permeability. In addition, severalexcreted periplasmic proteins have been separated from the culturemedium by antibody precipitation or SDS-polyacrylamide gelelectrophoresis in order to characterize these “periplasmic leaky”mutants. See, for example, Anderson et al., J. Bacteriol., 140(2):351-358 (1979) and Lazzaroni and Portalier, J. Bacteriol., 145 (3):1351-1358 (1981).

In a 10-kiloliter-scale process for recovery of IGF-I polypeptide (Hartet al., Bio/Technology, 12: 1113 (1994)), the authors attempted thetypical isolation procedure involving a mechanical cell breakage stepfollowed by a centrifugation step to recover the solids. The resultswere disappointing in that almost 40% of the total product was lost tothe supernatant after three passes through the Gaulin homogenizer. Hartet al., Bio/Technology 12: 1113 (1994). See FIG. 2 herein. Productrecovery was not significantly improved even when the classicaltechniques of EDTA and HEW-lysozyme additions were employed.

While HEW-lysozyme is the only practical commercial lysozyme forlarge-scale processes, lysozyme is expressed by bacteriophages uponinfection of host cells. Lysis of E. coli, a natural host forbacteriophages, for example the T4 phages, requires the action of twogene products: e and t. Gene e encodes a lysozyme (called T4-lysozymefor the T4 phage) that has been identified as a muramidase (Tsugita andInouye, J. Biol. Chem., 243: 391 (1968)), while gene t seems to berequired for lysis, but does not appear to have lysozyme activity. Genet is required for the cessation of cellular metabolism that occursduring lysis (Mukai et al., Vir., 33: 398 (1967)) and is believed todegrade or alter the cytoplasmic membrane, thus allowing gene product eto reach the periplasm and gain access to the cell wall (Josslin, Vir.,40: 719 (1970)). Phage are formed by gene t-mutants, but lysis of the E.coli host does not occur except by addition of chloroform (Josslin,supra). Wild-type T4-lysozyme activity is first detected about eightminutes after T4 infection at 37° C., and it increases through the restof the infection, even if lysis inhibition is induced. In the absence ofsecondary adsorption, cells infected by gene e mutants shut down progenyproduction and metabolism at the normal time, but do not lyse (MolecularGenetics of Bacteriophage T4, J. D. Karam, ed. in chief (AmericanSociety for Microbiology, Washington D.C., ASM Press, 1994), p. 398).

Recovery of insoluble IGF-I using T4-lysozyme was disclosed on Oct. 28,1997 at the “Separation Technology VII meeting entitled ‘Separations forClean Production’” in Davos, Switzerland, sponsored by the EngineeringFoundation.

For controlling cost of goods and minimizing process time, there is acontinuing need for increasing the total recovery of insolubleheterologous polypeptides contained in refractile particles from theperiplasmic space of prokaryotes. Further, there is a need for culturingof E. coli cells to high cell densities as an important factor forachieving efficient recombinant heterologous polypeptide production.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a process for recoveringrefractile particles containing a heterologous polypeptide frombacterial periplasm in which the polypeptide is insoluble comprising:

(a) culturing bacterial cells, which cells comprise nucleic acidencoding phage lysozyme, nucleic acid encoding the heterologouspolypeptide, a signal sequence for secretion of the heterologouspolypeptide, and separate promoters for each of the nucleic acidencoding the phage lysozyme and the nucleic acid encoding theheterologous polypeptide, wherein the promoter for the heterologouspolypeptide is inducible and the promoter for the phage lysozyme iseither a promoter with low basal expression or an inducible promoter,wherein in the absence of induction the promoter for the phage lysozymeis a promoter with low basal expression, the culturing being underconditions whereby when an inducer is added, expression of the nucleicacid encoding the phage lysozyme is induced after about 50% or more ofthe heterologous polypeptide has accumulated, and under conditionswhereby the heterologous' polypeptide is secreted into the periplasm ofthe bacteria as an aggregate and the phage lysozyme accumulates in acytoplasmic compartment;

(b) disrupting the cells mechanically to release the phage lysozyme soas to release refractile particles from cellular matrix; and

(c) recovering the released refractile particles from the periplasm,whereby chloroform is not used in any step of the process, and whereinthe recovery step minimizes co-recovery of cellular debris with thereleased refractile particles.

It was found in cell recovery that mechanical breakage by itself is notsufficient for efficient release of the insoluble polypeptide inaggregate form such as refractile particles and that HEW-lysozyme doesnot work well. Coordinated expression of nucleic acid encoding phagelysozyme with nucleic acid encoding the polypeptide of interest providesa highly effective method for releasing insoluble refractile particlesfrom the entanglement with the peptidoglycan layer. When the phagelysozyme gene is cloned behind a tightly-controlled promoter, forexample, the pBAD promoter (also referred to as the ara promoter),cytoplasmic accumulation of phage lysozyme may be induced by theaddition of an inducer (such as arabinose) at an appropriate time nearthe end of fermentation. By placing the nucleic acid expression ofheterologous polypeptide and phage lysozyme under separate promotercontrol, one can independently regulate their production duringfermentation. Without a signal sequence, the accumulated phage lysozymeis tightly locked up in the cytoplasmic compartment. Upon mechanicaldisruption of the cells, phage lysozyme is released to degrade thepeptidoglycan layer. Furthermore, the optimal pH for T4-phage-lysozymeactivity, which is a preferred embodiment, is about 7.3, which is aboutthe neutral pH of most typical harvest broths.

The induction of the gene encoding the bacteriophage lysozyme afterexpression of the nucleic acid encoding the heterologous polypeptideresults in a significant increase in the amount of insolubleheterologous polypeptide recovered from the periplasm of bacteria aftermechanical cell disruption. The phage lysozyme is trapped in thecytoplasmic compartment during fermentation until release by suchdisruption. Besides product yield, the success of a recovery process isjudged by the ease of operation, the process flow, the turn-around time,as well as the operation cost. The present invention alleviates severalif not all these bottlenecks encountered in the large-scale recoveryprocess.

The process herein also allows use of phage lysozyme at high celldensity and increased scale. At high density, even partial leakiness ofexpression could have disastrous results. Further, it would not beexpected that induction at the end of a long fermentation process andafter substantial product accumulation would produce enough of the phagelysozyme to be effective. The present process does not pose problems athigh cell densities such as increased viscosity and excessive foamingduring the fermentation process. The examples herein demonstrate thatthe process of this invention enables the attainment of high celldensity, effective induction and action of the system, and theprocessing of lysates derived from high-density cultures. Additionally,at least certain embodiments of the process herein require lessmechanical disruption of the cells, leading to less large-scaleprocessing time than with conventional processing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic diagram of how a protein product is disposedin the periplasm once made in the cytoplasm, that is, it forms anaggregate, proteolyzed fragment, or folded soluble protein.

FIG. 2 depicts IGF-I aggregate recovery from the supernatant and pelletby the typical isolation procedure involving mechanical cell disruptionfollowed by centrifugation, after three passes through the Gaulinhomogenizer.

FIG. 3 depicts plasmids employed in the construction of pIGFLysAra, usedto produce IGF-I, namely pJJ115, pT4lystacII, pLBIGF57, and pT4LysAra.

FIG. 4 depicts a schematic of the co-expression of T4-lysozyme, apreferred phage lysozyme, and IGF-I nucleic acid in accordance with anexample of this invention.

FIG. 5 depicts a graph of various parameters of the fermentation processfor IGF-I using co-expression of T4-lysozyme nucleic acid with IGF-Inucleic acid for a fermentation experiment disclosed herein.

FIG. 6 shows the time course of optical density for seven IGF-Ifermentation experiments entitled SI1613 (solid diamonds), SI1609(downward solid triangles), SI1599 (solid large circles), SI1608 (opentriangles), SI1610 (checked squares), SI1554 (upward solid triangles),and SI1547 (solid small squares). The experiment identification key is:

Experiment # Production Organism Test Condition SI1613 45F8/pLBIGF57Control organism, no arabinose induction SI1609 45F8/pIGFLysAra Minusarabinose induction control SI1599 45F8/pIGFLysAra 0.1% arabinoseinduction at 32 hrs SI1608 45F8/pIGFLysAra 1% arabinose induction at 36hrs SI1610 45F8/pIGFLysAra 1% arabinose induction at 32 hrs SI155445F8/pIGFLysAra 1% arabinose induction at 32 hrs SI1547 45F8/pIGFLysAra0.1% arabinose induction at 24 hrs

FIG. 7 shows the time course of oxygen uptake rate for the sevenfermentation experiments described for FIG. 6.

FIG. 8 shows the time course of oxygen transfer profile (KLA) for theseven fermentation experiments described for FIG. 6.

FIG. 9 shows the time course of IGF-I polypeptide accumulation for fivefermentation experiments described for FIG. 6, i.e., -T4-lysozymecontrol (diamonds), -arabinose control (downward solid triangles), 1%ara at 36 hours (upward open triangles), 1% ara at 32 hours (quarteredsquares), and 0.1% ara at 24 hours (circles).

FIG. 10 shows the total schematic for the steps of this invention forproducing IGF-I from culturing by use of induction of T4-lysozymenucleic acid expression to isolation of IGF-I aggregates involvingmechanical disruption and centrifugation.

FIGS. 11A and 11B respectively show percent total IGF-I productrecovered and percent insoluble product recovered when 0.1% arabinose at32 hours (dark bars), or 1% arabinose at 36 hours (stippled bars) isused for 0, 1, and 2 hours of incubation at 37° C. The product isrecovered by centrifugation at 5000 rpm×30 minutes in a SORVAL™centrifuge using a GSA rotor.

FIG. 12 shows the percent of cell-associated IGF-I recovered for 0 or 2hours of incubation for (1) no lysozyme, (2 and 3) HEW-lysozyme+EDTAaddition, (4 and 5) T4-lysozyme induced by 0.1% arabinose (32 hr), and(6) T4-lysozyme induced by 1% arabinose (36 hr).

FIG. 13 shows the plasmid construction of pJJ153, which is used toexpress T4-lysozyme-encoding nucleic acid from a plasmid separate fromthat used for DNase (pLS20) or VEGF (pVEGF171) expression, where pJJ153is prepared from pACYC177 and pT4LysAra.

FIG. 14 discloses the forward and complementary nucleotide sequences(SEQ ID NOS:1 and 2, respectively) and the amino acid sequence (SEQ IDNO:3) for the STII signal sequence and DNase used for construction ofpLS20.

FIG. 15 discloses the plasmid construction of pLS20, a DNase expressionvector, from pTF111, pLS18, and pBR322.

FIG. 16 shows the gel electrophoresis results in 10-20% TRICINE™pre-cast gels (Novex) of VEGF broth induced for T4-lysozymeco-expression, where the left-most lane is molecular weight standards,the next lane is whole broth, the third lane is pellet passed throughthe MICROFLUIDIZER® mechanical disruption device (Microfluidics, Inc.,Newton, Mass.), the fourth lane is supernatant, the fifth lane is theresuspended pellet from broth after three passes through theMICROFLUIDIZER® device (M3P) plus 5 mM final concentration EDTA (LE) at37° C. for one hour, the sixth lane is M3P plus LE at 37° C. for twohours, and the seventh (right-most) lane is M3P plus LE at 37° C. fortwo hours with room-temperature incubation for 18 hours, for a total of20 hours.

FIG. 17 shows the 10-20% TRICINE™ gel electrophoresis results of DNasebroth induced for T4-lysozyme co-expression, where the left-most lane ismolecular weight standards, the next lane is whole broth, the third laneis pellet passed through the MICROFLUIDIZER® device, the fourth lane issupernatant, the fifth lane is the resuspended pellet from broth afterthree passes through the MICROFLUIDIZER® device (M3P) plus 5 mM finalconcentration EDTA at 37° C. for two hours, the sixth lane is theresuspended pellet from broth after one pass through the MICROFLUIDIZER®device (M1P) plus 5 mM final concentration EDTA at 37° C. for two hours,and the seventh (right-most) lane is M3P with no EDTA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Definitions

As used herein, “phage lysozyme” refers to a cytoplasmic enzyme thatfacilitates lysis of phage-infected bacterial cells, thereby releasingreplicated phage particles. The lysozyme may be from any bacteriophagesource, including T7, T4, lambda, and mu bacteriophages. The preferredsuch lysozyme herein is T4-lysozyme.

As used herein, “T4-lysozyme”, or “E protein”, refers to a cytoplasmicmuramidase that facilitates lysis of T4 phage-infected bacterial cells,thereby releasing replicated phage particles (Tsugita and Inouye, J.Mol. Biol., 37: 201-12 (1968); Tsugita and Inouye, J. Biol. Chem., 243:391-97 (1968)). It is encoded by gene e of T4 bacteriophage andhydrolyzes bonds between N-acetylglucosamine and N-acetylmuramic acidresidues in the rigid peptidoglycan layer of the E. coli cell envelope.The enzyme is a single polypeptide chain of a molecular weight of 18.3kd. It is approximately 250-fold more active than HEW-lysozyme againstbacterial peptidoglycan (Matthews et al., J. Mol. Biol., 147: 545-558(1981)). The optimal pH for T4-lysozyme enzyme activity is 7.3, versus 9for HEW-lysozyme. (The Worthington Manual; pp 219-221).

As used herein, the phrase “agent that disrupts the outer cell wall” ofbacteria refers to a molecule that increases permeability of the outercell wall of bacteria, such as chelating agents, e.g., EDTA, andzwitterions.

As used herein, the term “bacteria” refers to any bacterium thatproduces proteins that are transported to the periplasmic space. Theterm “non-temperature-sensitive bacteria” refers to any bacterium thatis not significantly sensitive to temperature changes. Generally, thebacteria, whether gram positive or gram negative, has lysozyme-encodinggene expression under control so that the gene is only expressed nearthe end of the fermentation, a preferred embodiment, or expressed at alow level during fermentation. Also, preferably the bacteria do notcontain a temperature-sensitive phage repressor gene. Hence, thepreferred non-temperature-sensitive bacteria herein are distinguishedfrom bacteria containing defective temperature-sensitive lysogens suchas lambda prophage that lack genes necessary for replication orstructural protein assembly so that functional phage cannot be produced.The most preferred bacteria herein are gram-negative bacteria and/orbacteria that are non-temperature sensitive.

As used herein, “a time sufficient to release the polypeptide containedin the periplasm” refers to an amount of time sufficient to allow thelysozyme to digest the peptidoglycan to a sufficient degree to releasethe periplasmic aggregate or polypeptide.

As used herein, “signal sequence” or “signal polypeptide” refers to apeptide that can be used to secrete the heterologous polypeptide intothe periplasm of the bacteria. The signal for the heterologouspolypeptide may be homologous to the bacteria, or they may beheterologous, including signals native to the polypeptide being producedin the bacteria.

As used herein, “inducible” promoters are promoters that directtranscription at an increased or decreased rate upon binding of atranscription factor.

As used herein, a “promoter with low basal expression” or a“low-basal-level-expression promoter” is a promoter that is slightlyleaky, i.e., it provides a sufficiently low basal expression level so asnot to affect cell growth or product accumulation and provides asufficiently low level of promotion not to result in premature celllysis.

“Transcription factors” as used herein include any factors that can bindto a regulatory or control region of a promoter and thereby effecttranscription. The synthesis or the promoter-binding ability of atranscription factor within the host cell can be controlled by exposingthe host to an “inducer” or removing a “repressor” from the host cellmedium. Accordingly, to regulate expression of an inducible promoter, aninducer is added or a repressor removed from the growth medium of thehost cell.

As used herein, the phrase “induce expression” means to increase theamount of transcription from specific genes by exposure of the cellscontaining such genes to an effector or inducer.

An “inducer” is a chemical or physical agent which, when given to apopulation of cells, will increase the amount of transcription fromspecific genes. These are usually small molecules whose effects arespecific to particular operons or groups of genes, and can includesugars, alcohol, metal ions, hormones, heat, cold, and the like. Forexample, isopropylthio-β-galactoside (IPTG) and lactose are inducers ofthe tacII promoter, and L-arabinose is a suitable inducer of thearabinose promoter.

A “repressor” is a factor that directly or indirectly leads to cessationof promoter action or decreases promoter action. One example of arepressor is phosphate. As the repressor phosphate is depleted from themedium, the alkaline phosphatase (AP) promoter is induced.

As used herein, “polypeptide” or “polypeptide of interest” refersgenerally to peptides and proteins having more than about ten aminoacids. Those that are secreted to the periplasm of the bacteria mayassociate with the bacterial cell wall. The recovery of thesepolypeptides from bacterial periplasm improves with the co-expression ofphage lysozyme. The polypeptides are “heterologous,” i.e., foreign tothe host cell being utilized, such as a human protein produced by E.coli. The polypeptide is produced as an insoluble aggregate in theperiplasmic space.

Examples of mammalian polypeptides include molecules such as, e.g.,renin, a growth hormone, including human growth hormone; bovine growthhormone; growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; lipoproteins; α1-antitrypsin; insulin A-chain;insulin B-chain; proinsulin; thrombopoietin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; clotting factorssuch as factor VIIIC, factor IX, tissue factor, and von Willebrandsfactor; anti-clotting factors such as Protein C; atrial naturieticfactor; lung surfactant; a plasminogen activator, such as urokinase orhuman urine or tissue-type plasminogen activator (t-PA); bombesin;

thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; a serum albumin such as human serum albumin;mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors;integrin; protein A or D; rheumatoid factors; a neurotrophic factor suchas brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β;cardiotrophins (cardiac hypertrophy factor) such as cardiotrophin-1(CT-1); platelet-derived growth factor (PDGF); fibroblast growth factorsuch as aFGF and bFGF; epidermal growth factor (EGF); transforminggrowth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1,TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II(IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growthfactor binding proteins; CD proteins such as CD-3, CD-4, CD-8, andCD-19; erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; anti-HER-2antibody; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing receptors;addressins; regulatory proteins; antibodies; and fragments of any of theabove-listed polypeptides.

The preferred exogenous polypeptides of interest are mammalianpolypeptides, most preferably human polypeptides. Examples of suchmammalian polypeptides include t-PA, VEGF, gp120, DNase, IGF-I, IGF-II,brain IGF-I, growth hormone, relaxin chains, growth hormone releasingfactor, insulin chains or pro-insulin, urokinase, immunotoxins,neurotrophins, and antigens. Particularly preferred mammalianpolypeptides include, e.g., IGF-I, DNase, or VEGF, most preferablyIGF-I.

As used herein, “IGF-I” refers to insulin-like growth factor from anyspecies, including bovine, ovine, porcine, equine, and preferably human,in native-sequence or in variant form and recombinantly produced. In apreferred method, the IGF-I is cloned and its DNA expressed, e.g., bythe process described in EP 128,733 published Dec. 19, 1984.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably-linked coding sequence in a particularhost organism. The control sequences that are suitable for bacteriainclude a promoter such as the alkaline phosphatase promoter, optionallyan operator sequence, and a ribosome-binding site.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading frame. Linking isaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, the synthetic oligonucleotide adaptors or linkers are usedin accordance with conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

B. Modes for Carrying Out the Invention

The present process addresses accumulation of insoluble aggregates inthe bacterial periplasm and recovery of refractile particles containingan insoluble heterologous polypeptide therefrom. Chloroform is not usedin any of the basic three steps of the process.

In the first step of this process, the bacterial cells, preferablynon-temperature-sensitive bacterial cells, are cultured so as to expressthe nucleic acid encoding the polypeptide in the periplasmic space. Thecells contain nucleic acid encoding a phage lysozyme, preferablyT4-lysozyme, nucleic acid encoding the heterologous polypeptide, asignal sequence for secretion of the heterologous polypeptide, andseparate promoters for each of the nucleic acid encoding the lysozymeand the nucleic acid encoding the heterologous polypeptide. Typically,the expression elements are introduced into the cells by transformationtherein, but they may also be integrated into the genome or chromosomeof the host bacterial cells along with their promoter regions. Further,the polypeptide-encoding nucleic acid and the phage-lysozyme-encodingnucleic acid may be contained on separate plasmids used to transform thecells or on one single plasmid.

In the process herein, induction of the promoters is preferred; however,the process also contemplates the use of a promoter for the lysozymethat is a promoter with low basal expression (slightly leaky), whereinno induction is carried out. This type of promoter has a leakiness thatis low enough not to result in premature cell lysis and results in asufficiently low basal expression level so as not to affect cell growthor product accumulation.

The culturing is carried out in a manner such that expression of thenucleic acid encoding the lysozyme, when induced, commences after about50% or more of the heterologous polypeptide has accumulated, and underconditions whereby the heterologous polypeptide is secreted into theperiplasm of the bacteria and the phage lysozyme accumulates in thecytoplasmic compartment.

The promoters for this process must be different, such that thenucleic-acid-encoded heterologous polypeptide expression is inducedbefore expression of nucleic-acid-encoded phage lysozyme or at a muchhigher level, when the promoters are inducible. While the promoters maybe any suitable promoters for this purpose, preferably, the promotersfor the lysozyme and polypeptide are, respectively, arabinose promoterand alkaline phosphatase promoter. Alternatively, thecompartmentalization of the phage lysozyme may allow for the use of apromoter with low basal expression for expression of the nucleic acidencoding phage lysozyme. If a promoter with low basal expression isemployed, such as arabinose as opposed to tac or trp promoter, then anactive step of induction is not required.

The induction of expression of the nucleic acid encoding the phagelysozyme is preferably carried out by adding an inducer to the culturemedium. While, in this respect, the inducers for the promoters may beadded in any amount, preferably if the inducer is arabinose, it is addedin an amount of about 0-1% by weight, and if inducer is added, 0.1-1% byweight.

While the signal sequence may be any sequence, including the nativesignal sequence, if the polypeptide is IGF-I, preferably the signalpeptide is lamB.

The culturing step takes place under conditions of high cell density,that is, generally at a cell density of about 15 to 150 g dryweight/liter, preferably at least about 40, more preferably about40-150, most preferably about 40 to 100. In optical density, 120 OD550(A₅₅₀) is about 50 g dry wt./liter. In addition, the culturing can beaccomplished using any scale, even very large scales of 100,000 liters.Preferably, the scale is about 100 liters or greater, more preferably atleast about 500 liters, and most preferably from about 500 liters to100,000 liters.

In the process described above, the bacterial cells may be transformedwith one or two expression vectors containing the nucleic acid encodingthe phage lysozyme and the nucleic acid encoding the heterologouspolypeptide. In one such embodiment, the bacterial cells are transformedwith two vectors respectively containing the nucleic acid encoding thephage lysozyme and the nucleic acid encoding the heterologouspolypeptide. Such two-plasmid system allows for the moderation of genecopy number. In another such embodiment, the nucleic acid encoding thephage lysozyme and the nucleic acid encoding the heterologouspolypeptide are contained on one vector with which the bacterial cellsare transformed. Alternatively, the nucleic acid encoding the phagelysozyme and/or polypeptide, along with the respective promoter(s)therefor, is integrated into the host chromosome.

In the first step, the heterologous nucleic acid (e.g., cDNA or genomicDNA) is suitably inserted into a replicable vector for expression in thebacterium under the control of a suitable promoter for bacteria. Manyvectors are available for this purpose, and selection of the appropriatevector will depend mainly on the size of the nucleic acid to be insertedinto the vector and the particular host cell to be transformed with thevector. Each vector contains various components depending on itsfunction (amplification of DNA or expression of DNA) and the particularhost cell with which it is compatible. The vector components forbacterial transformation will include a signal sequence for thepolypeptide and will also include an inducible promoter for thepolypeptide and an inducible promoter or a non-inducible one with lowbasal expression for the phage lysozyme. They also generally include anorigin of replication and one or more marker genes.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with bacterial hosts. The vector ordinarily carries areplication site, as well as marking sequences that are capable ofproviding phenotypic selection in transformed cells. For example, E.Coli is typically transformed using pBR322, a plasmid derived from an E.coli species. See, e.g., Bolivar et al., Gene, 2: 95 (1977). pBR322contains genes conferring ampicillin and tetracycline resistance andthus provides an easy means for identifying transformed cells. ThepBR322 plasmid, or other microbial plasmid or phage, also generallycontains, or is modified to contain, promoters that can be used by thebacterial organism for expression of the selectable marker genes.

The DNA encoding the polypeptide of interest herein contains a signalsequence, such as one at the N-terminus of the mature polypeptide. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the polypeptide DNA that is inserted into the vector. Theheterologous signal sequence selected should be one that is recognizedand processed (i.e., cleaved by a signal peptidase) by the host cell.For bacterial host cells that do not recognize and process the nativepolypeptide signal sequence, the signal sequence is substituted by abacterial signal sequence selected, for example, from the groupconsisting of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders.

Expression vectors contain a nucleic acid sequence that enables thevector to replicate in one or more selected host cells. Such sequencesare well known for a variety of bacteria. The origin of replication fromthe plasmid pBR322 is suitable for most Gram-negative bacteria.

Expression vectors also generally contain a selection gene, also termeda selectable marker. This gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli. One example of a selectionscheme utilizes a drug to arrest growth of a host cell. Those cells thatare successfully transformed with a heterologous gene produce a proteinconferring drug resistance and thus survive the selection regimen.

The expression vector for producing a heterologous polypeptide alsocontains an inducible promoter that is recognized by the host bacterialorganism and is operably linked to the nucleic acid encoding thepolypeptide of interest. It also contains a separate promoter, which maybe inducible or of low basal expression, operably linked to the nucleicacid encoding the phage lysozyme. Inducible promoters suitable for usewith bacterial hosts include the β-lactamase and lactose promotersystems (Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature,281: 544 (1979)), the arabinose promoter system, including the araBADpromoter (Guzman et al., J. Bacteriol., 174: 7716-7728 (1992); Guzman etal., J. Bacteriol., 177: 4121-4130 (1995); Siegele and Hu, Proc. Natl.Acad. Sci. USA, 94: 8168-8172 (1997)), the rhamnose promoter (Haldimannet al., J. Bacteriol., 180: 1277-1286 (1998)), the alkaline phosphatasepromoter, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 (1980) and EP 36,776), the P_(LtetO-1) and P_(lac/ara−1)promoters (Lutz and Bujard, Nucleic Acids Res., 25: 1203-1210 (1997)),and hybrid promoters such as the tac promoter (deBoer et al., Proc.Natl. Acad. Sci. USA, 80: 21-25 (1983)). However, other known bacterialinducible and low-basal-expression promoters are suitable. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to DNA encoding the polypeptide ofinterest or to the phage lysozyme gene (Siebenlist et al., Cell, 20: 269(1980)) using linkers or adaptors to supply any required restrictionsites. If a strong and highly leaky promoter, such as the trp promoter,is used, it is generally used only for expression of the nucleic acidencoding the heterologous polypeptide and not forphage-lysozyme-encoding nucleic acid. The tac and P_(L) promoters couldbe used for either, but not both the polypeptide and phage lysozyme, butare not preferred. Preferred are the alkaline phosphatase promoter forthe product and the arabinose promoter for the phage lysozyme.

Promoters for use in bacterial systems also generally contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding thepolypeptide of interest. The promoter can be removed from the bacterialsource DNA by restriction enzyme digestion and inserted into the vectorcontaining the desired DNA. The phoA promoter can be removed from thebacterial-source DNA by restriction enzyme digestion and inserted intothe vector containing the desired DNA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) or other strains, and successful transformants are selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Sanger et al., Proc. Natl.Acad. Sci. USA, 74: 5463-5467 (1977) or Messing et al., Nucleic AcidsRes., 9: 309 (1981), or by the method of Maxam et al., Methods inEnzymology, 65: 499 (1980).

Suitable bacteria for this purpose include archaebacteria andeubacteria, especially eubacteria, more preferably Gram-negativebacteria, and most preferably Enterobacteriaceae. Examples of usefulbacteria include Escherichia, Enterobacter, Azotobacter, Erwinia,Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia,Shigella, Rhizobia, Vitreoscilla, and Paracoccus. Suitable E. coli hostsinclude E. coli W3110 (ATCC 27,325), E. coli 294 (ATCC 31,446), E. coliB, and E. coli X1776 (ATCC 31,537). These examples are illustrativerather than limiting. Mutant cells of any of the above-mentionedbacteria may also be employed. It is, of course, necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well-knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon.

E. Coli strain W3110 is a preferred host because it is a common hoststrain for recombinant DNA product fermentations. Preferably, the hostcell should secrete minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins, with examples of such hosts including E. coli W3110strain 1A2, which has the complete genotype tonAΔ (also known as ΔfhuA);E. coli W3110 strain 9E4, which has the complete genotype tonAΔ ptr3; E.coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotypetonAΔ ptr3 phoAΔE15 Δ(argF-lac)169 ompTΔ degP41kan^(r) ; E. coli W3110strain 37D6, which has the complete genotype tonAΔ ptr3 phoAΔE15Δ(argF-lac)169 ompTΔ degP41kan^(r) rbs7Δ ilvG; E. coli W3110 strain40B4, which is strain 37D6 with a non-kanamycin resistant degP deletionmutation; E. coli W3110 strain 33D3, which has the complete genotypetonA ptr3 lacIq LacL8 ompT degP kan^(r) ; E. coli W3110 strain 36F8,which has the complete genotype tonA phoA Δ(argF-lac) ptr3 degP kan^(R)ilvG+, and is temperature resistant at 37° C.; E. coli W3110 strain45F8, which has the complete genotype fhuA(tonA) Δ(argF-lac) ptr3degP41(kanS) Δ omp Δ(nmpc-fepE) ilvG+ phoA+ phoS*(T10Y); E. coli W3110strain 43E7, which has the complete genotype fhuA(tonA) Δ(argF-lac) ptr3degP41(kanS) ΔompTΔ(nmpc-fepE) ilvG+ phoA+; and an E. coli strain havingthe mutant periplasmic protease(s) disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990.

Host cells are transformed with the above-described expression vector(s)of this invention and cultured in conventional nutrient media modifiedas appropriate for inducing the various promoters if induction iscarried out.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or as chromosomalintegration. Depending on the host cell used, transformation is doneusing standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride, as described in section 1.82 ofSambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989), is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO, as described in Chungand Miller, Nucleic Acids Res., 16: 3580 (1988). Yet another method isthe use of the technique termed electroporation.

Bacterial cells used to produce the polypeptide of interest described inthis invention are cultured in suitable media in which the promoters canbe induced as described generally, e.g., in Sambrook et al., supra.

Any other necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrations,introduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. The pH of the medium may be any pH fromabout 5-9, depending mainly on the host organism.

For induction, typically the cells are cultured until a certain opticaldensity is achieved, e.g., a A₅₅₀ of about 80-100, at which pointinduction is initiated (e.g., by addition of an inducer, by depletion ofa repressor, suppressor, or medium component, etc.), to induceexpression of the gene encoding the heterologous polypeptide. When about50% or more of the polypeptide has accumulated (as determined, e.g., bythe optical density reaching a target amount observed in the past tocorrelate with the desired polypeptide accumulation, e.g., a A₅₅₀ ofabout 120-140), induction of the promoter is effected for the phagelysozyme. The induction typically takes place at a point in timepost-inoculation about 75-90%, preferably about 80-90%, of the totalfermentation process time, as determined from prior experience andassays. For example, induction of the promoter may take place at fromabout 30 hours, preferably 32 hours, up to about 36 hourspost-inoculation of a 40-hour fermentation process.

Gene expression may be measured in a sample directly, for example, byconventional northern blotting to quantitate the transcription of mRNA(Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)). Variouslabels may be employed, most commonly radioisotopes, particularly ³²PHowever, other techniques may also be employed, such as usingbiotin-modified nucleotides for introduction into a polynucleotide. Thebiotin then serves as the site for binding to avidin or antibodies,which may be labeled with a wide variety of labels, such asradionuclides, fluorescers, enzymes, or the like.

For accumulation of an expressed gene product, the host cell is culturedunder conditions sufficient for accumulation of the gene product. Suchconditions include, e.g., temperature, nutrient, and cell-densityconditions that permit protein expression and accumulation by the cell.Moreover, such conditions are those under which the cell can performbasic cellular functions of transcription, translation, and passage ofproteins from one cellular compartment to another for the secretedproteins, as are known to those skilled in the art.

After accumulation of the heterologous polypeptide in the periplasm, thecells are disrupted mechanically, or lysed, to release the phagelysozyme, which in turn acts to release refractile particles containingthe polypeptide from the cellular matrix, or cell wall. In a preferredembodiment, after disruption the cells are incubated for a period oftime sufficient to release the heterologous polypeptide contained in theperiplasm. This period of time will depend, for example, on the type ofpolypeptide being recovered and the temperature involved, but preferablywill range from about 1 to 24 hours, more preferably 2 to 24 hours, andmost preferably 2 to 3 hours. If there is overdigestion with the enzyme,the improvement in recovery of product will not be as great.

In a third step, the refractile particles released from the cellularmatrix are recovered from the periplasm, in a manner that minimizesco-recovery of particulate cellular debris with the particles. Therecovery may be done by any means, but preferably comprises sedimentingretractile particles containing the heterologous polypeptide orcollecting supernatant containing soluble product. An example ofsedimentation is centrifugation. In this case, the recovery preferablytakes place in the presence of an agent that disrupts the outer cellwall to increase permeability and allows more aggregated product to berecovered. Examples of such agents include a chelating agent such asEDTA or a zwitterion such as, for example, a dipolar ionic detergentsuch as ZWITTERGENT 316™ detergent. See Stabel et al., supra. Mostpreferably, the recovery takes place in the presence of EDTA.

If centrifugation is used for recovery, the relative centrifugal force(RCF) applied is an important factor. The RCF is adjusted to minimizeco-sedimentation of cellular debris with the refractile particlesreleased from the cell wall at lysis. The specific RCF used for thispurpose will vary with, for example, the type of product to berecovered, but preferably is at least about 3000×g, more preferablyabout 3500-6000×g, and most preferably about 4000-6000×g.

The duration of centrifugation will depend on several factors. Thesedimentation rate will depend upon, e.g., the size, shape, and densityof the refractile particle and the density and viscosity of the fluid.The sedimentation time for solids will depend, e.g., on thesedimentation distance and rate. It is reasonable to expect that thecontinuous disc-stack centrifuges would work well for the recovery ofthe released heterologous polypeptide aggregates or for the removal ofcellular debris at large scale, since these centrifuges can process athigh fluid velocities because of their relatively large centrifugalforce and the relatively small sedimentation distance.

The heterologous polypeptide in aggregate form may then be furtherpurified to obtain preparations that are substantially homogeneous as tothe polypeptide of interest. In a preferred embodiment, the aggregatedpolypeptide is isolated, followed by a simultaneous solubilization andrefolding of the polypeptide, as disclosed in U.S. Pat. No. 5,288,931.

The following procedures are exemplary of suitable purificationprocedures for the heterologous polypeptide once it is released from thecells: fractionation on immunoaffinity or ion-exchange columns; ethanolprecipitation; reverse-phase HPLC; chromatography on silica or on acation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; and gel filtration using, for example, SEPHADEX™G-75.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all patent and scientific referencescited in the specification are expressly incorporated herein byreference.

Example I

IGF-I and T4-Lysozyme Nucleic Acid Co-Expression Background:

IGF-I was selected as a first protein for evaluation of refractileparticle recovery due to large-scale needs. For this evaluation, astrategy was mapped out involving genetic manipulation of the hostorganisms to improve the release of the refractile particles fromcell-wall structures.

Materials & Methods:

pIGFLysAra Plasmid Construction: In pIGFLysAra, the IGF-I encodingsequence has a lamB signal sequence for secretion into the periplasm,and was placed behind the alkaline phosphatase promoter (AP). TheT4-lysozyme gene was placed behind the ara promoter for cytoplasmicaccumulation of the gene product.

Details of the construction of the original plasmid, pT4lystacII, havebeen described in Gene, 38: 259-264 (1985). Intermediate plasmids weremade to move the T4-lysozyme gene behind the ara promoter. Subsequently,the ara promoter-T4-lysozyme gene cassette was inserted into the IGF-Iplasmid (pLBIGF57), resulting in pIGFLysAra, a single plasmid for theco-expression of nucleic acid encoding IGF-I and T4-lysozyme (FIG. 3).

The plasmid pLBIGF57 was constructed from a basic backbone of pBR322.The transcriptional and translational sequences required for theexpression of nucleic acid encoding IGF-I were provided by the phoApromoter and trp Shine-Dalgarno. Secretion of the protein was directedby a TIR (translation-initiation region) variant of the lamB signalsequence. This TIR variant does not alter the primary amino acidsequence of the lamB signal; however, silent nucleotide sequence changesresult, in this particular variant, in an increased level of translatedprotein.

The details of pLBIGF57 construction follow. A codon library of the lamBsignal sequence was constructed to screen for translational initiationregion (TIR) variants of differing strength. Specifically, the thirdposition of codons 3 to 7 of the lamB signal sequence was varied. Thisdesign conserved the wild-type amino acid sequence and yet allowed fordivergence within the nucleotide sequence.

LamB TIR variants were selected covering an approximate 10-fold activityrange. Specifically, lamB TIR variant #57 provides an approximately 1.8fold stronger TIR than the wild-type lamB codons based on the phoAactivity assay.

The vector fragment for the construction of pLBIGF57 was generated bydigesting pBK131Ran with XbaI and SphI. This XbaI-SphI vector containsthe phoA promoter and trp Shine-Dalgarno sequences. The coding sequencesfor IGF-I and the lambda t_(o) transcription terminator were isolatedfrom pBKIGF-2B (U.S. Pat. No. 5,342,763) following digestion withNcoI-SphI. The lamB signal sequence fragment was isolated frompLBPhoTBK#57 (TIR variant #57; generated as described above) followingdigestion with XbaI-NcoI. These three fragments were then ligatedtogether to construct pLBIGF57.

pJJ115 was constructed as follows. The ClaI/AlwNI fragment from pBR322was inserted into ClaI/AlwNI-digested pBAD18 (Guzman et al., J.Bacteriol., 177: 4121-4130 (1995)) to produce pJJ70. One round ofsite-directed mutagenesis was then performed, changing HindIII to StuIto obtain pJJ75. A second round of site-directed mutagenesis was done tochange MluI to SacII, to produce pJJ76. Then XbaI/HindIII fragments frompJJ76 and pBKIGF-2B were ligated to obtain pJJ115. A schematic of theplasmid constructions is shown in FIG. 3.

FIG. 4 depicts a schematic of the co-expression of T4-lysozyme and IGF-Inucleic acid in accordance with this example.

Bacterial Strains and Growth Conditions: Most experiments were carriedout with strain 45F8 (E. coli W3110 fhuA(tonA)Δ(argF-lac) ptr3 degP41(kanS) ΔompTΔ(nmpc-fepE) ilvG+ phoA+ phoS*(T10Y)). Competent cells of45F8 were transformed with pIGFLysAra using the standard procedure.Transformants were picked after growth on an LB plate containing 50μg/mL carbenicillin (LB+CARB50™), streak-purified and grown in LB brothwith 50 μg/mL CARB50™ in a 37° C. shaker/incubator before being testedin the fermentor.

For comparison, the IGF-I plasmid without the ara promoter-T4-lysozymeexpression elements, pLBIGF57, replaced pIGFLysAra in controlexperiments conducted under similar conditions. pLBIGF57 confers bothcarbenicillin and tetracycline resistance to the production host andallows 45F8/pLBIGF57 to grow in the presence of either antibiotic.

Fermentation Process The fermentation medium composition andexperimental protocol used for the co-expression of nucleic acidencoding IGF-I and T4-lysozyme were similar to those of the scaled-downhigh-metabolic rate, high-yield 10-kiloliter IGF-I process. Briefly, ashake flask seed culture of 45F8/pIGFLysAra was used to inoculate therich production medium. The composition of the medium (with thequantities of each component utilized per liter of initial medium) isdescribed below:

Ingredient Quantity/L Glucose* 200-500 g Ammonium Sulfate 2-10 g SodiumPhosphate, Monobasic Dihydrate 1-5 g Potassium Phosphate, Dibasic 1-5 gSodium Citrate, Dihydrate 0.55 g Potassium Chloride 0.55 g MagnesiumSulfate, Heptahydrate 0.5-5 g PLURONIC ™ Polyol, L61 0.1-5 mL FerricChloride, Heptahydrate 10-100 mg Zinc Sulfate, Heptahydrate 0.1-10 mgCobalt Chloride, Hexahydrate 0.1-10 mg Sodium Molybdate, Dihydrate0.1-10 mg Cupric Sulfate, Pentahydrate 0.1-10 mg Boric Acid 0.1-10 mgManganese Sulfate, Monohydrate 0.1-10 mg Hydrochloric Acid 10-100 mgTetracycline 4-30 mg Yeast Extract* 5-25 g NZ Amine AS* 5-25 gMethionine* 0-5 g Ammonium Hydroxide as required to control pH SulfuricAcid as required to control *A portion of the glucose, yeast extract, NZAmine AS, and methionine is added to the medium initially, with theremainder being fed throughout the fermentation.

The fermentation was a fed-batch process with fermentation parametersset as follow:

Agitation: Initially at 800 RPM, increased to 1000 RPM at 8 OD Aeration:15.0 slpm pH control: 7.3 Temp. : 37° C. Back pressure: 0.7 bar Glucosefeed: computer-controlled using an algorithm which regulates the growthrate at approximately 95% of the maximum early in the fermentation andwhich then controls the dissolved oxygen concentration (DO₂) at 30% ofair saturation after the DO₂ drops to 30%. Complex nitrogen feed:constant feed rate of 0.5 mL/min throughout the fermentation experimentFermentation Experiment 40 hours Duration:

The timing of arabinose addition ranged from 24 hr to 36 hr. Bolusadditions of 0.1% to 1% (final concentration) arabinose were tested tohelp define the induction strength necessary for producing the mostpreferred amounts of T4-lysozyme for better product recovery at thecentrifugation step. A schematic of the fermentation process is shown inFIG. 5.

Recovery of Refractile Particle from Harvested Broth: Broth harvested atthe end of fermentation was either processed soon after or storedbriefly at 4° C. prior to use. The test protocol used involved fourprocess steps:

I. Break cells open by multiple passes through the MICROFLUIDIZER®mechanical disruption device (Microfluidics, Inc., Newton, Mass.) torelease T4-lysozyme from the cytoplasmic compartment of cells. Thisoperation was carried out at room temperature instead of 4° C. toaccelerate the enzymatic degradation of peptidoglycan by T4-lysozyme.

II. Add 1M EDTA to the broth passed through the MICROFLUIDIZER® device(microfluidized broth) to bring the final concentration of EDTA to 5 mM.EDTA chelates the divalent cations and disrupts the outer cell surfacestructure. This makes the peptidoglycan layer inside unbroken cellsaccessible to degradation by T4-lysozyme and weakens the cell wall topromote cell lysis.

III. Hold the lysate at room temperature or incubate at 37° C. forfurther degradation of cell wall. This step was introduced to simulatethe longer process times associated with the larger-scale process and toevaluate product stability in the lysate.

IV. Recover refractile particles from the lysate by centrifugation.Bench-scale centrifugation in a SORVALL™ GSA rotor at two speeds (5000rpm & 4000 rpm; equivalent to RCF's of 4056×g and 2603×g at rmax,respectively) was used to collect the solids as pellets.

An additional step to wash the pellet with buffer was investigated topurify the refractile particles further. This step would remove thelysate entrained by the pellet and minimize the amount of contaminatingE. coli proteins in the refractile particle preparations.

Aliquots of the treated broth were saved after three passes through theMICROFLUIDIZER® device and after each subsequent treatment step. Sampleswere evaluated for the percent of the whole broth recovered in thepellet by centrifugation (% pellet). Dry weights of samples weredetermined by measuring the net sample weights before and after dryingfor a minimum of two days in a vacuum oven controlled at approximately60-70° C. Ratio of the dried weight to that of the wet weight for thesample was expressed as percentage and reported as the % dry weight forthe sample analyzed. The amount of product present in the samples wasanalyzed by a HPLC reverse-phase method. Product recovery efficiency wascalculated by expressing the amount of product recovered by the processstep as a percent of the total product present in the starting material,the fermentation broth at harvest. To evaluate the quality of therefractile particle recovered, the protein profile of refractileparticles resuspended in 50 mM Tris, pH 7.5 w/5 mM EDTA to the originalvolume was compared to that of the harvest broth sample by gelelectrophoresis in 10-20% TRICINE™ pre-cast gels (Novex). Bydensitometry, the percentage of the total protein represented by theproduct could be calculated.

Results & Discussion:

Fermentation and IGF-I Production:

The initial growth rate of 45F8/pIGFLysAra showed no significantdifference from that of the 45F8/pLBIGF57 control in the fermentationprocess described. As expected, broth cell density reached over 120OD550 after 14 to 16 hrs. Cell density subsequently peaked and remainedsteady to the end of the experiment (FIG. 6).

Typical respiration rates were obtained for both fermentations. Therewas a slight, but continuous drop in oxygen uptake rate (OUR,mmoles/L-min) after 32 to 34 hrs post inoculation in most experiments(FIG. 7). As this loss in respiration was seen with both experimentaland control experiments, the phenomenon was probably not caused byT4-lysozyme nucleic acid co-expression.

To avoid competition for the host's synthetic capacity, T4-lysozymenucleic acid expression, regulated by the controllable pBAD (arabinose)promoter, was to be induced by the addition of the arabinose afterproduct accumulation stopped. However, there were indications of leakyexpression of nucleic acid encoding T4-lysozyme. A slight leakage ofT4-lysozyme from the cytoplasmic compartment was also suggested. Themost direct evidence came from light microscopy images of cells carryingthe T4-lysozyme nucleic acid expression elements. Broth samples takenprior to induction showed a small number of round-shaped cells mixedwith the typical rod-shaped cells expected for E. coli. No round-shapedcells were visible in the control broth. Without being limited to anyone theory, most likely, in these round cells, T4-lysozyme-encodingnucleic acid was expressed prematurely and leaked into the periplasm,compromising the peptidoglycan layer to weaken the mechanical supportrequired for maintenance of the rod shape.

The notion of T4-lysozyme leakage was further supported by theobservation of increased broth viscosity in harvested broth of45F8/pIGFLysAra upon extended storage at 4° C. The same was not seenwith fermentation broth of 45F8/pLBIGF57. Some cell walls weakened byT4-lysozyme leakage apparently were no longer able to contain thecytoplasmic turgor pressure. Release of DNA and other cytoplasmiccontents by the lysed cells resulted in an increase in fluid viscosity.Also, based on the profile of oxygen transfer during fermentation byplotting kla (a measurement of oxygen transfer capacity) againstexperiment time, there appeared to be a decline in kla that correlatedwith the arabinose induction in experiments co-expressing nucleic acidencoding T4-lysozyme (FIG. 8). The loss in oxygen transfer could beexplained by an increase in broth viscosity caused byT4-lysozyme-induced cell lysis.

Except for one case, the co-expression of T4-lysozyme nucleic acid hadno impact on IGF-I accumulation (FIG. 9). When 0.1% arabinose was addedto the culture 24 hrs after inoculation, the accumulation of IGF-Iceased soon after the arabinose addition. Under control conditions,IGF-I synthesis begins when the medium is depleted of free phosphates(typically around 12-14 hrs). By about 30-32 hrs post inoculation, thecells have accumulated the majority of the IGF-I found in the harvestedbroth. By this time, IGF-1-specific productivity drops offsignificantly. Without being limited to any one theory, one possibleexplanation for the yield loss associated with the 0.1% arabinoseinduction at 24 hrs might be that T4-lysozyme synthesis competed againstIGF-I synthesis for the protein synthesis machinery remaining in thecells. When the arabinose addition was made after 32 hrs, the impact ofT4-lysozyme nucleic acid expression became insignificant since onlyminimal amounts of IGF-I were being made at this time.

FIG. 10 shows a schematic of the process shown by this Example, withdownstream processing indicated as an inventory step or protein foldingafter the centrifugation (recovery) step.

FIGS. 11A and 11B show respectively the percent of total productrecovered and of insoluble product recovered after 0 to 2 hours ofincubation at 37° C. for 0.1% ara at 32 hours and 1% ara at 36 hours. Itis clear that percent product recovered increases with incubation, and1% ara at 36 hours increased recovery at all incubation conditionsexcept for percent of insoluble product recovered after two hours, wherethe 0.1% ara showed increased product recovery.

T4-lysozyme-encoding nucleic acid expression was qualitativelydemonstrated by 10-20% TRICINE™ gel electrophoresis. Samples taken fromexperiments with earlier or stronger arabinose inductions (SI1547induced at 24 hrs w/0.1% arabinose; SI1610 induced at 32 hr w/1%) showedin their protein profile a new protein band with the expected molecularweight of 18.3 kilodaltons for T4-lysozyme. Cells at these stages duringfermentation were relatively healthy and capable of limited synthesis ofT4-lysozyme upon induction. When induction was delayed to 36 hrs, noT4-lysozyme band was visible by gel analysis.

Under the fermentation and induction conditions used, T4-lysozyme wasnot expected to accumulate in substantial quantities. With the highspecific activity of T4-lysozyme against the E. coli peptidoglycan, asmall amount of T4-lysozyme is expected to be sufficient for the desireddegradation of the cell wall.

Recovery of Refractile Particle from Harvested Broth:

For insoluble material, the refractile particle recovery process takesadvantage of the stable nature of proteins sequestered as insolubleaggregates. The bench-scale cell disruption process was performed atroom temperature in the absence of temperature control. Cell lysateexiting from the mechanical device was slightly warm to the touch. Underthese conditions, the breakdown of the peptidoglycan matrix by theT4-lysozyme began immediately upon release of T4-lysozyme and was notretarded by the cooling typically employed to minimize proteindenaturation and degradation by proteases.

As broth was sent through the MICROFLUIDIZER® device, there appeared tobe an increase in viscosity immediately after the first passage. Therewere small amounts of gelatinous mass within the lysed broth thatdisappeared with time or after additional passes. After the second pass,the viscosity of the lysed broth appeared to have decreased. Photostaken of IGF-I cells induced for T4-lysozyme revealed good cell breakageefficiency. However, the released retractile particles appeared to haveclumped together in some samples.

With the typical working volume at the start of the refractile particlerecovery process of two liters, the bench-scale step of passing thebroth through the MICROFLUIDIZER® device usually required less than anhour for completing three passes of the treated broth. For simulation oflarger-scale processing that would take longer, a hold (incubation)step, also serving as additional reaction time for the lysozymaldegradation of peptidoglycan, was introduced prior to the centrifugationstep. In this study, hold times of 0, 2-3 and 24 hours were tested.Solids and product recoveries by centrifugation at two different speedsfrom these treated broths are summarized in Tables 1 and 2.

After centrifugation at approximately 4000×g (GSA rotor at 5000 rpm) for30 min (Table 1), the control broth with no T4-lysozyme nucleic acidexpression elements on the plasmid (45F8/pLBIGF57 as productionorganism) yielded a much smaller pellet by wet weight than any of thebroths with some level of T4-lysozyme nucleic acid co-expression.Similar results were obtained for the percent dry weight represented bythe pellet. The percentage of cell-associated IGF-I product recovered inthe pellet differed significantly between these experimental conditions.When centrifugation was performed immediately after passage through theMICROFLUIDIZER® device, only 26-28% of the cell-associated IGF-I in thestarting material was recovered in the pellet for the control(45F8/pLBIGF57) As much as three times that amount was recovered fromthe 45F8/pIGFLysAra culture grown without arabinose induction; it islikely there was leaky expression of nucleic acid encoding T4-lysozyme.With a 2-3-hour incubation prior to centrifugation, greater than 90% oftotal cell-associated IGF-I was found in the pellet. Further extensionof the incubation time from 2-3 hours to 24 hours had little effect.

Solids recovery from treated broths by centrifugation was also tested atlower speed, approximately 2600×g for 30 min (Table 2). The resultingpellets collected were significantly smaller. The lower g-force wasgenerally insufficient to sediment the solids suspended in the lysedbroth, and therefore product recovery was poor.

The recovery of solids by centrifugation is governed by the physicalproperties of both the solids and the fluid in the suspension.

The settling velocity of the particles is defined by the followingequation:

Settling Velocity of the Particle (v_(c))

$v_{c} = \frac{\left( {\rho_{p} - \rho_{m}} \right)d^{2}\omega^{2}r}{18\mu}$where ρ_(p)=particle density, ρ_(m)=fluid density, μ=fluid viscosity,ω=angular velocity, d=particle diameter, and r=radial distance.

According to this equation, while the diameter of the particle is themost important factor governing the settling velocity of the particle,the settling velocity of the particle is directly proportional to thedifference between the densities of the particle and the fluid and isinversely proportional to the viscosity of the fluid containing theparticle.

The refractile particles of IGF-I have been shown by electron microscopyto be large, dense globules. Other work had suggested that the densityof IGF-I refractile particles was higher than that of the E. coli cellwall. These properties of the IGF-I refractile particles should makethem amenable to efficient recovery by centrifugation. However, if therefractile particles have not been cleanly released from the cell wallat the cell lysis step, the refractile particles trapped by intact cellsand cell wall debris will behave as composites and co-sediment withother solids. After digestion of the peptidoglycan layer withT4-lysozyme, if successfully freed from cellular matrix, the settlingvelocity of the freed IGF-I refractile particles, now being solelydetermined by its intrinsic physical properties, is higher than that ofE. coli cell wall fragments. Centrifugation parameters may bemanipulated to minimize co-sedimentation of cell debris with the freedretractile particles. The success of this genetic solution involvingT4-lysozyme nucleic acid co-expression is evident from the greater than95% product recovery shown in Table 1.

TABLE 1 Recovery of Solids from Microfluidized Broth (Three Passes) byBench-Scale Centrifugation SORVALL ™ GSA rotor, 5000 RPM for 30 min(4056 × g at r_(max)) at 4-15° C. Hold % Cell-Asso- ExperimentProduction Induction Time % Pellet % Dry ciated IGF-I No. OrganismConditions (hr)^(a) Recovered^(b) Weight^(c) Recovery (TypicalNon-microfluidized Control Whole Broth) 18-23 8.0-8.9 100 SI161345F8/pLBIGF57 Control organism, no 0 4.7 ND 26.0 ara SI162445F8/pLBIGF57 Control organism, no 0 4.8 1.31 28.2 ara SI160945F8/pIGFLysAra Minus ara control 0 8.7 2.23 76.3 SI1610 45F8/pIGFLysAra1% ara @ 32 hrs 0 5.3 1.58 47.6 SI1624 45F8/pLBIGF57 Control organism,no 2-3 6.8 1.57 33.4 ara SI1609 45F8/pIGFLysAra Minus ara control 2-311.8 2.49 95.8 SI1599 45F8/pIGFLysAra 0.1% ara @ 32 hrs 2-3 11.4 2.7298.3 SI1610 45F8/pIGFLysAra 1% ara @ 32 hrs 2-3 7.9 1.95 59.7 SI160245F8/pIGFLysAra 1% ara @ 36 hrs 2-3 10.2 2.26 90.2 SI1613 45F8/pLBIGF57Control organism, no 24 7.8 1.93 35.8 ara SI1609 45F8/pIGFLysAra Minusara control 24 12.3 2.75 90.8 SI1610 45F8/pIGFLysAra 1% ara @ 32 hrs 2410.8 2.51 81.1 Footnotes to Table 1: ^(a)Microfluidized broth was heldat 37° C. for the first three hours and transferred to room temperaturefor the remaining time prior to centrifugation to help determine thebest exposure of lysed broth to released T4-lysozyme required forefficient refractile particle recovery at the centrifugation step.^(b)Percent pellet recovered is the percent of the whole broth recoveredin the pellet by centrifugation. ^(c)Percent dry weight is the ratio ×100 of the dried weight to the initial wet weight for the sample(fermentation broth), where the dry weight is determined as describedabove.

TABLE 2 Recovery of Solids from Microfluidized Broth (Three Passes) byBench-Scale Centrifugation SORVALL ™ GSA rotor, 4000 RPM for 30 mm (2603× g at r_(max)) at 4-15° C. % Cell- Hold Associated ExperimentProduction Induction Time % Pellet % Dry IGF-I No. Organism Conditions(hr)^(a) Recovered^(b) Weight^(c) Recovery (Typical Non-microfluidizedControl Whole Broth) 18-20 8.0-8.9 100 SI1613 45F8/pLBIGF57 Controlorganism, no 0 2.4 ND 17.7 ara SI1553 45F8/pLBIGF57 Minus ara control 13.2 1.14 >36.0 SI1599 45F8/pIGFLysAra 0.1% ara @ 32 hrs 1 6.0 1.41 19.5SI1554 45F8/pIGFLysAra 1% ara @ 32 hrs 1 3.8 1.18 26.3 SI154745F8/pLBIGF57 0.1% ara @ 24 hrs 1 2.1 0.90 15.5 SI1613 45F8/pIGFLysAraControl organism, no 24 5.7 1.51 24.9 ara SI1609 45F8/pIGFLysAra Minusara control 24 13.1 ND ND SI1610 45F8/pIGFLysAra 1% ara @ 32 hrs 24 11.6ND ND ^(a)Microfluidized broth was held at 37° C. for the first threehours and transferred to room temperature for the remaining time priorto centrifugation to help determine the best exposure of lysed broth toreleased T4-lysozyme required for efficient refractile particle recoveryat the centrifugation step. ^(b)Percent pellel: recovered is the percentof the whole broth recovered in the pellet by centrifugation.^(c)Percent dry weight is the ratio × 100 of the dried weight to theinitial wet weight for the sample (fermentation broth), where the dryweight is determined as described above.T-4 Lysozyme vs. HEW-Lysozyme:

The effectiveness of endogenous T4-lysozyme versus the exogenousHEW-lysozyme on the recovery of IGF-I refractile particles was comparedin Table 3, and shown in a bar graph in FIG. 12. As a replacement forT4-lysozyme, HEW-lysozyme was added to 0.2 mg/mL (final concentration)(previously found to be the optimal concentration for cell lysis) tomicrofluidized control broth (45F8/pLBIGF57 that had its pH adjusted to9.0). The resultant broth was carried through the remaining recoverysteps alongside the T4-lysozyme-containing broths. As shown in Table 3,addition of HEW-lysozyme increased the % of cell-associated IGF-Irecovered from 28-33% for the control case to 42-52%. However, comparedto the greater-than-90% product recovery for broths with T4-lysozymenucleic acid co-expression, HEW-lysozyme treatment was clearly andunexpectedly much inferior.

The protein profile of the recovered IGF-I refractile particle wasexamined by 10-20% TRICINE™ gel electrophoresis. The pellets collectedby centrifugation were resuspended in buffer to the original volume,allowing for direct comparison to the control samples. Gel analysis ofthe various β-mercaptoethanol-reduced refractile particle samples showeda significant clean-up compared to the whole broth control. In general,there was little qualitative difference between samples held for a widerange of times for extended enzymatic degradation of peptidoglycan bythe T4-lysozyme. The T4-lysozyme was highly efficient in releasing therefractile particles from cells.

TABLE 3 Effectiveness of Endogenous T4-lysozyme vs ExogenousHEW-Lysozyme on IGF-I Refractile Particle Recovery from MicrofluidizedBroth^(a) (Three Passes) by Bench Scale Centrifugation SORVALL ™ GSArotor, 5000 RPM for 30 mm (4056 × g at r_(max)) at 4-15° C. Hold % CellExperiment Production Induction Time % Pellet % Dry Associated IGF-I No.Organism Conditions (hr)^(b) Recovered^(c) Weight^(d) Recovery (TypicalNon-microfluidized Control Whole Broth) 18-23 8.0-8.9 100 SI162445F8/pLBIGF57 Control organism, 0 4.8 1.31 28.2 no ara No lysozyme, no 26.8 1.57 33.4 EDTA 0.2 mg/mL HEW- 0 6.2 1.62 42.9 lysozyme plus EDTA 27.0 1.70 51.9 SI1599 45F8/pIGFLysAra 0.1% ara @ 32 hrs 0 4.6 1.26 38.0T4-lysozyme plus EDTA 2 11.4 2.72 98.3 SI1602 45P8/pIGFLysAra 1% ara @36 hrs 0 8.4 1.95 88.3 T4-lysozyme plus EDTA 2 10.2 2.26 90.2 ^(a)Thetypical starting working volume was 2 L of harvest broth and the step ofpassing through the MICROFLUIDIZER ® device usually required 30 to 45min. ^(b)Microfluidized broth was held at 37° C. for degradation ofpeptidoglycan by released T4-lysozyme or added HEW-lysozyme prior torecovering refractile particles at the centrifugation step. ^(c)and^(d)See footnotes b and c of Table 1.

Example II VEGF or DNase and T4-Lysozyme Nucleic Acid Co-ExpressionBackground

It was important to determine if the T4-lysozyme nucleic acidco-expression technology had general application across other processesinvolving refractile particles. E.-coli-produced VEGF (a 21kD protein)and DNase (a 31.9-kilodalton protein) were two additional products knownto accumulate in the periplasmic space as retractile particles andtherefore suitable proteins for evaluation. It was difficult to predictif T4-lysozyme nucleic acid co-expression would bring similar benefitsto product recovery since it was not known if the physical properties ofthe retractile particles of VEGF and DNase differ significantly fromthat of the IGF-I retractile particles.

For efficient evaluation of the T4-lysozyme nucleic acid co-expressionapproach in multiple processes, a separate plasmid for the expression ofnucleic acid encoding T4-lysozyme, pJJ153, was constructed. It was usedin the co-transformation of the appropriate host organism along with theproduct plasmid encoding either VEGF (pVEGF171) or DNase (pLS20).

Materials and Methods:

pJJ153 Plasmid Construction: The construction of pJJ153 (a pACYC177derivative that is compatible with pBR322 vectors) is shown in FIG. 13.The ClaI/AlwNI fragment from pBR322 was inserted intoClaI/AlwNI-digested pBAD18 (Guzman et al., supra) to produce pJJ70. Oneround of site-directed mutagenesis was then performed, changing HindIIIto StuI to obtain pJJ75. A second round of site-directed mutagenesis wasdone to change MluI to SacII, to produce pJJ76. Then XbaI/HindIIIfragments from pJJ76 and from pBKIGF2B were ligated, and XbaI/HindIIIfragments from this ligation product and from a T4-lysozyme/tac plasmidwere ligated to produce pT4LysAra. Then BamHI (filled in)/ScaI-digestedpACYC177 was ligated with ClaI/HindIII (both ends filled in)-digestedpT4LysAra to produce pJJ153. The maps for pACYC177, pT4LysAra, andpJJ153 are shown in FIG. 13.pVEGF171 Plasmid Construction: The construction of pVEGF171 is asfollows: pVEGF171 is a derivative of the previously published VEGF165plasmid with a TIR relative strength of 3 (Simmons et al., NatureBiotechnology, 14:629-634 (1996)). In this plasmid the mature VEGFcoding sequence (Leung et al., Science, 246:1306-1309 (1989)) ispreceded by that of the STII signal sequence (Picken et al., Infect.Immun., 42:269-275 (1983); Lee et al., Infect. Immun., 42:264-268(1983)) to provide for secretion of VEGF into the E. coli periplasmicspace. Transcription of the heterologous gene is provided for by thealkaline phosphatase promoter (Kikuchi et al., Nucleic Acids Res.,9:5671-5678 (1981)), and translation initiation is controlled by thesilent codon changes in the STII signal sequence as noted (Simmons etal., Nature Biotechnology, 14:629-634 (1996)). The only change inpVEGF171 from the above-described VEGF165 plasmid is the addition of theλ to transcriptional terminator (Scholtissek et al., Nucleic Acids Res.,15:3185 (1987)) just downstream of the VEGF termination codon, followedby the fully restored tetracycline resistance gene of pBR322 (Bolivar etal., Gene, 2:95-113 (1977)).pLS20 Plasmid Construction: The plasmid pLS20 is a pBR322 (Sutcliffe,Cold Spring harbor Symp. Quant. Biol., 43: 77-90 (1978)))-based plasmiddesigned for the expression of DNase in E. coli. The transcriptional andtranslational sequences required for the expression of DNase areprovided by the alkaline phosphatase promoter, the trp Shine-Dalgarnoand the STII Shine-Dalgarno, as described for the plasmid phGH1 (Changet al., Gene, 55: 189-196 (1987)). Secretion of the protein from thecytoplasm to the periplasmic space is directed by the STII signalsequence (Picken et al., Infect. Immun., 42: 269-275 (1983)). Downstreamof the DNase coding sequence is the tetracycline resistance gene Theforward and complementary nucleotide sequences (SEQ ID NOS: 1 and 2,respectively) and the amino acid sequence (SEQ ID NO:3) for the STIIsignal sequence and DNase are provided in FIG. 14.

The construction of pLS20 is as follows: The vector fragment for theconstruction of pLS20 was generated by digesting pTF111 with NsiI-BamHIand isolating the largest fragment. The plasmid pTF111 is a derivativeof phGH1 (Chang et al., supra), and an identical vector would have beengenerated had phGH1 been used in place of pTF111. The second fragmentrequired for this construction was isolated from pLS18 followingdigestion with NsiI-HindIII. The HindIII site of this fragment wasblunted by treatment with DNA Polymerase I (Klenow). The coding sequencefor DNase is contained within this approximately 790-bp fragment. Thefinal fragment necessary for the ligation was generated by digestingpBR322 with EcoRI-BamHI. The EcoRI site of this fragment was blunted bytreatment with DNA Polymerase I (Klenow). This fragment of approximately380 bp contained the tetracycline-resistance promoter and the 5′ end ofthe tetracycline-resistance coding sequence. These three fragments wereligated together as illustrated in FIG. 15 to construct pLS20.

Bacterial strains and growth conditions: Both the VEGF and DNasefermentations conducted for this example used strain 43E7 as productionhost (E. coli W3110 fhuAtonA Δ(argF-lac)169 ptr3 degP41(kanS)ΔompTΔ(nmpc-fepE) ilvG+ phoAΔE15). Competent cells of 43E7 weretransformed with pJJ153 and either pVEGF171 or pLS20 using standardprocedures. Transformants were picked from LB plates containing 20 μg/mLtetracycline and 50 μg/mL kanamycin (LB+Tet20+Kan50), streak-purified,and grown in LB broth with 20 μg/mL tetracycline and 50 μg/mL kanamycinin a 37° C. or 30° C. shaker/incubator before being stored in DMSO at−80° C.

For control conditions, the host 43E7 transformed with either pVEGF171or pLS20 alone was used for the VEGF or DNase processes, respectively.

VEGF Fermentation Process: The fermentation medium composition andexperimental protocol used for the co-expression of VEGF and T4-lysozymenucleic acids were as follows. A shake flask seed culture of43E7/pVEGF171 or 43E7/pVEGF171/pJJ153 was used to inoculate the richselective production medium, and the fermentation parameters were set asfollows:

Agitation: 1000 RPM Aeration: 10.0 slpm pH control : 7.2 Temp.: 37° C.Back pressure: 0.3 bar Glucose feed: computer controlled, initiallyusing an algorithm to control the culture near its maximum growth ratewithout glucose overfeeding. When the DO₂ reaches 30% of air saturation,the algorithm adjusts the glucose feed rate to maintain the DO₂ at 30%.Fermentation experiment 40 hours duration:

1% arabinose additions were made at 26 hr or 32 hr post inoculation forthe induction of T4-lysozyme nucleic acid expression.

DNase Fermentation Process: A shake flask seed culture of 43E7/pLS20 or43E7/pLS20/pJJ153 grown in LB plus the appropriate antibiotics was usedto inoculate the rich selective production medium in the fermentor, andthe fermentation parameters were set as follow:

Agitation: 1000 RPM Aeration: 10.0 slpm pH control: 7.2 Temp. : 30° C.Back pressure: 0.3 bar Glucose feed: computer controlled, initiallyusing an algorithm to control the culture near its maximum growth ratewithout glucose overfeeding. When the DO₂ reaches 30% of air saturation,the algorithm adjusts the glucose feed rate to maintain the DO₂ at 30%.Complex nitrogen feed: 0.25 mL/min starting at 20 OD

1% arabinose addition was made at 24 hr or 32 hr post inoculation forthe induction of T4-lysozyme nucleic acid expression. Broth washarvested at 32 to 36 hrs post inoculation for refractile particlerecovery evaluation.

Recovery of Refractile Particles from Harvested VEGF and DNase Broth:

Broth harvested at the end of the fermentations was either processedfresh or stored briefly at 4° C. prior to use. The test protocoldescribed earlier for IGF-I refractile particle recovery evaluation wasused for the evaluation of refractile particle recovery from the VEGFand DNase broths.

Results:

Fermentation Process:

Growth of the VEGF and the DNase cultures induced for T4-lysozymenucleic acid co-expression (directed by pJJ153) looked similar to therespective minus-pJJ153 controls. All of the VEGF and DNasefermentations showed an aggressive drop in OUR (mmoles/L-min) late inthe fermentation process. At the same time, optical density (OD550) alsodeclined significantly. Such dramatic loss in cell density was notobserved for IGF-I, a fed-batch process with a single plasmid for theco-expression of nucleic acid encoding the product and T4-lysozyme.

In experiment SRPVF2, 1% arabinose was added at 32 hr post inoculationfor the induction of T4-lysozyme nucleic acid co-expression, well afterthe respiration rate of the cells had started to decline. T4-lysozymeproduction would likely be minimal. When the final broth sample wasexamined under phase-contrast microscopy, it was observed that asubstantial percentage of the cell population had lost the rod shapetypical of intact healthy cells. Again, this observation was in linewith that made with IGF-I, suggesting a low-level leakage of T4-lysozymeinto the periplasmic compartment. That the broth viscosity appearednormal suggested the absence of significant cell lysis at this time.Similar results were obtained in another experiment (SRPVF5) in whicharabinose was added earlier (1% arabinose added at 26 hrs).

Like the VEGF fermentations, the respiration rate for the DNase culturedeclined late in the fermentation process at about 25-28 hrs postinoculation. Arabinose additions made at 24 hrs for induction ofT4-lysozyme nucleic acid co-expression did not alter the on-set of theOUR loss. There were noticeable differences between the DNase process onthe one hand, and the VEGF and IGF-I processes on the other. The celllysis found in the harvested DNase broth that had been induced with a 1%arabinose addition made at 24 hrs was more severe than with the controlwithout arabinose addition. When the induced culture was examined underthe microscope, there appeared to be more weakened round-shaped cellsand ghost cells that had lost their cellular contents than healthyintact cells.

Recovery of Refractile Particle from Harvested VEGF Broth:

The VEGF harvest broth showed some increase in broth viscosity after asingle passage through the MICROFLUIDIZER® device, with transientvisible lumpiness in the mechanically-disrupted broth. With anadditional passage through the MICROFLUIDIZER® device, the brothviscosity was dramatically reduced. When mechanically-disrupted brothswere centrifuged immediately after a single pass or two passes throughthe MICROFLUIDIZER® device, the resulting pellets appeared very spongyand tended to decant off with the supernatant if not carefully handled.Similar observations had been made with IGF-I control broth in the past.

Solids were recovered by centrifugation after three passes through theMICROFLUIDIZER® device. The size of the pellets recovered from brothsco-expressing nucleic acid encoding T4-lysozyme and held for 2 hrs at37° C. was similar to that obtained from control whole broth that is notmechanically disrupted and not passed through the MICROFLUIDIZER®device, indicating that, without being limited to any one theory, almostall of the solids were collected at the RCF of 4056×g and nodifferential sedimentation of VEGF aggregates from cell debris wasachieved.

Qualitative assessment of product recovery efficiency was made by visualinspection of the intensity of the product band resolved by 10-20%TRICINE™ gel electrophoresis (see FIG. 16). Recovery of the VEGFaggregates appeared comparable across the various samples tested(including the lanes marked M3P/LE-1 hr (three passes plus 5 mM EDTAfinal concentration held at 37° C. for 1 hour), M3P/LE-2 hr (threepasses plus 5 mM EDTA final concentration held at 37° C. for 2 hours),and M3P/LE-20 hr (three passes plus 5 mM EDTA final concentration heldat 37° C. for 2 hours with room-temperature incubation for 18 hours).The gel profiles showed that a significant amount of the total proteinsoriginally present in the whole broth co-sedimented with the VEGFaggregates. Without being limited to any one theory, it is believed thatthe refractile particles of VEGF might not have been as freed ofcontaminating E. coli proteins as the IGF-I refractile particles, andthat the refractile particles of different products might differ intheir densities or sizes, depending on the underlying phenomena thatcaused the aggregation of the proteins. Additionally, without limitationto any one theory; it is believed that the refractile particles mightalso vary in the manner in which they associated with other E. coliproteins and therefore cause different amounts of contaminating proteinsto be entrapped in the product aggregates. Taken together, the resultssuggested that adjustments of the refractile particle recovery protocolwithin the guidelines set forth herein would maximize recovery ofdifferent refractile particles, depending on the polypeptide beingrecovered.

Recovery of Refractile Particles from Harvested DNase Broth:

During the processing of DNase broth for retractile particle recovery,the DNase pellets obtained by centrifugation after each of the threepasses or one pass through the MICROFLUIDIZER® device appeared to bemore compact than those of IGF-I or VEGF. The 10-20% TRICINE™electrophoresis gel profile in FIG. 17 showed good recovery of DNase forthe three process treatments. In fact, comparison of the lanes markedM3P (three passes plus 5 mM EDTA final concentration held at 37° C. for2 hours), M1P (one pass plus 5 mM EDTA final concentration held at 37°C. for 2 hours), and M3P; no EDTA (three passes without EDTA) revealedthat DNase requires only one pass through the MICROFLUIDIZER® device tobe recovered in good quantity. The comparison also shows that it ispreferred to add EDTA. These results illustrate the advantage of thisinvention in requiring less mechanical disruption of the cells so thatless large-scale processing time is required than with conventionalprocessing.

Conclusions:

Co-expression of nucleic acid encoding a phage lysozyme (such asT4-lysozyme) with nucleic acid encoding a heterologous polypeptide,either from an ara-promoter-regulated gene inserted into the productplasmid or directed by a similar ara-promoter-phage-lysozyme-genecassette in a second compatible plasmid, has been demonstrated toenhance recovery of heterologous polypeptide insoluble in the periplasmin accordance with the process of this invention. Under the conditionsemployed, the induction of expression of nucleic acid encoding phagelysozyme with arabinose addition did not negatively affect cell growthor product accumulation. Compared to HEW-lysozyme, the recovery ofperiplasmic refractile particles increased from approximately 50 to 90%with T4-lysozyme nucleic acid co-expression. The refractile particlerecovery protocol established is both simple and scalable.

1. A process for recovering refractile particles containing aheterologous polypeptide from bacterial periplasm in which thepolypeptide is insoluble comprising: (a) culturing bacterial cells,which cells comprise nucleic acid encoding phage lysozyme, nucleic acidencoding the heterologous polypeptide, a signal sequence for secretionof the heterologous polypeptide, and separate and different induciblepromoters for each of the nucleic acid encoding the phage lysozyme andthe nucleic acid encoding the heterologous polypeptide, whereby theheterologous polypeptide is secreted into the periplasm of the bacteriaas an aggregate and the phage lysozyme accumulates in the cytoplasmiccompartment, wherein expression of the nucleic acid encoding the phagelysozyme is induced by the addition of an inducer after about 50% ormore of the heterologous polypeptide has accumulated; (b) disrupting thecells mechanically to release the phage lysozyme so as to releaserefractile particles from cellular matrix; and (c) recovering thereleased refractile particles from the periplasm, whereby chloroform isnot used in any step of the process, and wherein the recovery stepminimizes co-recovery of cellular debris with the released refractileparticles.
 2. The process of claim 1 wherein the heterologouspolypeptide is a mammalian polypeptide.
 3. The process of claim 2wherein the mammalian polypeptide is a human polypeptide.
 4. The processof claim 3 wherein the human polypeptide is an insulin-like growthfactor (IGF), DNase, or vascular endothelial growth factor (VEGF). 5.The process of claim 4 wherein the human polypeptide is IGF-I.
 6. Theprocess of claim 5 wherein the promoters for the phage lysozyme andpolypeptide are, respectively, arabinose promoter and alkalinephosphatase promoter.
 7. The process of claim 6 wherein the inducer forarabinose is added in an amount of about 0-1% by weight.
 8. The processof claim 5 wherein the signal sequence is lamB.
 9. The process of claim1 wherein the bacterial cells are Gram-negative cells.
 10. The processof claim 9 wherein the bacterial cells are E. coli.
 11. The process ofclaim 1 wherein the bacterial cells are transformed with one or twoexpression vectors containing the nucleic acid encoding the phagelysozyme and the nucleic acid encoding the heterologous polypeptide. 12.The process of claim 11 wherein the bacterial cells are transformed withtwo vectors respectively containing the nucleic acid encoding the phagelysozyme and the nucleic acid encoding the heterologous polypeptide. 13.The process of claim 11 wherein the nucleic acid encoding the phagelysozyme and the nucleic acid encoding the heterologous polypeptide arecontained on one vector with which the bacterial cells are transformed.14. The process of claim 1 wherein after disruption the cells areincubated for a time sufficient to release the heterologous polypeptideaggregate contained in the periplasm.
 15. The process of claim 1 whereinthe recovery comprises sedimenting retractile particles containing theheterologous polypeptide.
 16. The process of claim 15 wherein therecovery takes place in the presence of an agent that disrupts the outercell wall of the bacterial cells.
 17. The process of claim 16 whereinthe agent is a chelating agent or zwitterion.
 18. The process of claim17 wherein the agent is EDTA.
 19. The process of claim 15 wherein thesedimentation is by centrifugation and is at a relative centrifugalforce of at least about 3000×g.
 20. The process of claim 1 wherein theculturing step takes place under conditions of a cell density of about40 to 150 g dry weight/liter.
 21. The process of claim 1 wherein thephage lysozyme is T4-lysozyme.
 22. The process of claim 1 wherein theculturing takes place at a scale of at least about 500 liters.
 23. Theprocess of claim 1 wherein the bacterial cells arenon-temperature-sensitive.
 24. The process of claim 1 wherein one ormore of the nucleic acids, including the promoter therefor, isintegrated into the genome of the bacterial cells.